Modified rnase h and detection of nucleic acid amplification

ABSTRACT

A reversibly modified ‘hot start’ RNase H enzyme composition is described for the improved CATACLEAVE™ probe detection of nucleic acid sequences in a test sample. A key feature of the enzyme composition is the ability to regulate the catalytic activity of the RNase H during the course of a reverse transcription-PCR cycle. Thus, RNase H activity can be initially suppressed to minimize degradation of RNA:DNA primer heteroduplexes prior to reverse transcription. After cDNA synthesis is complete, RNase H activity is induced to promote the cleavage and fluorescent detection of CATACLEAVE™ probes that anneal to target DNA sequences within the reverse transcriptase-PCR products. The inducible RNase H enzyme is amenable to high throughput applications requiring one step reverse transcriptase CATACLEAVE™ PCR in a single reaction mix.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/108,311 filed on May 16, 2011 (now allowed), which claimspriority from U.S. Provisional Patent Application No. 61/347,984, filedon May 25, 2010, the contents of which are hereby incorporated byreference in their entirety.

FIELD

The disclosure describes a modified RNAse H for improved real-timereverse transcriptase-PCR detection of RNA sequences.

BACKGROUND

One of the most widely used techniques to study gene expression exploitsfirst-strand cDNA of mRNA sequence(s) as a template for PCRamplification. The ability to measure the kinetics of a PCR reaction incombination with reverse transcriptase-PCR techniques promises tofacilitate the accurate and precise measurement of target RNA sequenceswith the requisite level of sensitivity. In particular, fluorescentdual-labeled hybridization probe technologies, such as the “CATACLEAVE™endonuclease assay (described in detail in U.S. Pat. No. 5,763,181; seeFIG. 1), permit the detection of reverse transcriptase-PCR amplificationin real-time. Detection of target sequences is achieved by including aCATACLEAVE™ probe in the amplification reaction together with RNAse H.The CATACLEAVE™ probe, which is complementary to a target sequencewithin the reverse transcriptase-PCR amplification product, has achimeric structure comprising an RNA sequence and a DNA sequence, and isflanked at its 5′ and 3′ ends by a detectable marker, for example FRETpair labeled DNA sequences. The proximity of the FRET pair's fluorescentlabel to the quencher precludes fluorescence of the intact probe. Uponannealing of the probe to the reverse transcriptase-PCR product a RNA:DNA duplex is generated that can be cleaved by RNAse H present in thereaction mixture. Cleavage within the RNA portion of the annealed proberesults in the separation of the fluorescent label from the quencher anda subsequent emission of fluorescence.

SUMMARY

A reversibly modified ‘hot start’ RNAse H enzyme composition isdescribed for improved CATACLEAVE™ probe detection of nucleic acidsequences in a test sample. A key feature of the enzyme composition isthe ability to regulate the catalytic activity of the RNAse H during thecourse of a reverse transcription-PCR cycle. Thus, RNAse H activity canbe initially suppressed to minimize degradation of RNA:DNA primerheteroduplexes prior to reverse transcription. After cDNA synthesis iscomplete, RNAse H activity is induced to promote the cleavage andfluorescent detection of CATACLEAVE™ probes that anneal to target DNAsequences within the reverse transcriptase-PCR products. The inducibleRNAse H enzyme is amenable to high throughput applications requiring onestep reverse transcriptase CATACLEAVE™ PCR in a single reaction mix.

In one embodiment, the invention includes a hot start enzyme compositioncomprising an enzyme having an inducible RNAse H activity. The enzymemay have a thermostable RNase H domain which can have at least 70%, 80%or 90%, 95% or 99% sequence identity to the amino acid sequence of SEQID NOs: 11, 12, 13 or 14.

The hot start composition can have a polymerase activity such as a DNApolymerase or reverse transcriptase activity. The RNAse H activity canbe heat-inducible or pH inducible.

In one embodiment, the enzyme with the inducible RNAse H activity can bePyrococcus furiosus RNase HII, Pyrococcus horikoshi RNase HII,Thermococcus litoralis RNase HI, Thermus thermophilus RNase HI, E. coliRNAse HI, or E. coli RNase HII.

The enzyme with an inducible RNAse H activity can be reversibly modifiedby a chemical modification. In an embodiment, the enzyme may bereversibly modified by acylation of an amino acid of the enzyme such aslysine or by reaction with formaldehyde having a concentration of about0.2 to about 1% (w/v).

In one embodiment, the hot start enzyme composition further comprises aligand, wherein the inducible RNAse H activity can be inhibited by theassociation of the enzyme with the ligand or induced by interferencewith the association of the enzyme with the ligand. The ligand can bethermolabile. The association between the enzyme and the ligand can benon-covalent.

The ligand can be a peptide, a nucleic acid or a small molecule having aK_(D) dissociation constant of 10⁻¹ M or less for the enzyme with theinducible RNAse H activity. In some embodiments, the ligand can bind tothe RNAse H domain or it can induce a conformational change in the RNAseH domain.

The ligand can be an antibody, an antibody fragment, an aptamer, or achelating agent.

The antibody fragment can be a Fab, a Fab′, a F(ab′)₂, a Fd, a singlechain Fv or scFv, a disulfide linked Fv, a V-NAR domain, a IgNar, anintrabody, an IgGDCH₂, a minibody, a F(ab′)₃, a tetrabody, a triabody, adiabody, a (scFv)₂, single-domain antibody, DVD-Ig, Fcab, mAb₂, or ascFv-Fc.

The RNAse H activity can be induced by (1) heating a solution containingthe enzyme to a temperature of about 90° C. or higher or (2) by loweringthe pH of a solution containing the enzyme to about 7.0 or lower. Thesolution can be a polymerase chain reaction sample comprising a targetnucleic acid sequence.

The association between the enzyme and the ligand can be covalent. Forexample, the ligand can be a cross-linking agent.

In one embodiment, the invention discloses a method of amplifying atarget sequence, having the steps of providing an amplification reactionmix having:

a sample comprising a target DNA sequence;

a hot start enzyme composition, comprising DNA polymerase and inducibleRNAse H activities;

a first primer comprising a sequence complementary to the 5′ end of thetarget nucleic acid sequence;

a second primer comprising a sequence complementary to the 3′ end of thetarget nucleic acid sequence; and

a probe which is coupled to a detectable label and having a compositioncapable of being cleaved by RNase H; and

subjecting the amplification reaction composition to at least oneamplification reaction to form at least one amplification product, and

measuring the detectable label of the resulting amplification product,wherein the amplification reaction comprises a step of heating thereaction composition to a temperature of about 90° C.

The step of heating the reaction composition can be conducted at about95° C.

The probe can be an oligonucleotide comprising one or more DNA sequenceportions and a RNA sequence portion, wherein the RNA portion is disposedbetween two DNA sequences in a way that the 3′ end and 5′ end of the RNAsequence are coupled to each of the two DNA sequences.

The heating step can induce the RNAse H activity by disrupting theassociation between the enzyme with the RNAse H activity and the ligand.The ligand can be thermolabile.

In another embodiment the invention discloses a method for detecting atarget ribonucleic acid sequence in a sample, having the steps ofproviding an amplification reaction composition comprising:

-   -   a sample containing a target RNA sequence,    -   a hot start enzyme composition comprising reverse transcriptase,        DNA polymerase and inducible RNAse H activities wherein the        RNAse H activity is inhibited by a ligand;    -   a probe sequence which contains a detectable label and comprises        a cleavage sequence of the RNase H;    -   a first primer comprising a sequence complementary to the 5′ end        of the target nucleic acid sequence; and    -   a second primer comprising a sequence complementary to the 3′        end of the target nucleic acid sequence;    -   initiating reverse transcription of the target RNA to form a        RNA: (NOTE: cDNA implies that the product is double stranded DNA        and not the reverse transcriptase product) DNA duplex;    -   heating the reaction composition to a temperature of about        90° C. or higher thereby activating the inducible RNAse H        activity to degrade the RNA moiety of the RNA: DNA duplex;    -   initiating at least one amplification reaction to form at least        one amplification product, and measuring the detectable label of        the resulting amplification products.

The probe can be an oligonucleotide comprising one or more DNA sequenceportions and a RNA sequence portion, wherein the RNA portion is disposedbetween two DNA sequences in a way that the 3′ end and 5′ end of the RNAsequence are coupled to each of the two DNA sequences. The targetribonucleic acid sequence can be a retrovirus. The step of heating thereaction composition can be conducted at about 95° C. The heating stepmay activate the inducible RNAse H activity. The ligand can bethermolabile.

In another embodiment, the invention describes a microarray having thehot start enzyme composition described herein.

In yet another embodiment, the invention discloses a method fordetecting target ribonucleic acid sequences in a plurality of samples,having the steps of providing a microarray having a plurality ofamplification reaction compositions each comprising:

-   -   a sample containing a target RNA sequence,    -   a hot start enzyme composition comprising reverse transcriptase,        DNA polymerase and inducible RNAse H activities wherein the        RNAse H activity is inhibited by a ligand;    -   a probe sequence which contains a detectable label and comprises        a cleavage sequence of the RNase H;    -   a first primer comprising a sequence complementary to the 5′ end        of the target nucleic acid sequence; and    -   a second primer comprising a sequence complementary to the 3′        end of the target nucleic acid sequence;    -   and for each amplification reaction composition in the        microarray:    -   initiating reverse transcription of the target RNA to form a        RNA: cDNA duplex, heating the reaction composition to a        temperature of about 90° C. or higher thereby activating the        inducible RNAse H activity to degrade the RNA moiety of the RNA:        cDNA duplex,    -   initiating at least one amplification reaction to form at least        one amplification product, and measuring the detectable label of        the resulting amplification product.

The target ribonucleic acid sequence can be a retrovirus. The step ofheating the reaction composition can be conducted at about 95° C. thatcan activate the inducible RNAse H activity. This ligand can bethermolabile.

In one embodiment, the invention discloses a kit having a microarray ofhot start compositions that may include an enzyme with reversetranscriptase activity or DNA polymerase activity.

The previously described embodiments have many advantages, includingusing a modified RNAse H enzyme to improve the sensitivity ofCATACLEAVE™ reverse transcription-PCR detection of RNA sequences. Theimproved detection method is fast, accurate and suitable for highthroughput applications. Convenient, user-friendly and reliablediagnostic kits are also described for the high throughput detection ofRNA sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The figures are not intended tolimit the scope of the teachings in any way.

FIG. 1 is a schematic representation of CATACLEAVE™ probe technology, asdescribed in U.S. Pat. No. 5,753,181,

FIG. 2 is a schematic representation of a method for real-timemonitoring nucleic acid amplification using a CATACLEAVE™ probe which isdegraded by an endonuclease,

FIG. 3 is a reaction scheme of acylation of RNase HII according to oneembodiment,

FIG. 4 are graphs showing the activity of the formaldehyde-treated RNaseHII, measured at 37° C. and 50° C. without activation and at 60° C.after 95° C. activation as described in Example 2,

FIGS. 5A and 5B are graphs depicting the performance (fluorescence inFIG. 5A, Cp valued in FIG. 5B) of the reactivated Pfu RNase HII whichhas been formaldehyde-treated,

FIGS. 6A and 6B are graphs depicting the activity of reversibly acylatedPfu RNase HII without reactivation (FIG. 6A) and with reactivation (FIG.6B),

FIGS. 7A and 7B are graphs depicting the activity of reactivated PfuRNase HII, which has been acylated, at pH 8.4 (FIG. 7A) and 8.7 (FIG.7B),

FIG. 8 is a graph depicting the endonuclease activity of untreated RNaseHII, and reversibly modified RNase HII, measured on HIV-1 genomic RNA,according to Example 6,

FIG. 9 depicts graphs showing the sensitivity of reversibly acylated PfuRNase HII for the detection of HIV-1 genomic RNA, as described inExample 7,

FIG. 10 depicts graphs showing the detection of Salmonella invA RNAusing unmodified Pfu RNase HII and reversibly acylated RNase HII, and

FIGS. 11A and 11B depict graphs showing a determination of RNase HIIactivity, as described in Example 1.

DETAILED DESCRIPTION

The practice of the embodiments described herein employs, unlessotherwise indicated, conventional molecular biological techniques withinthe skill of the art. Such techniques are well known to the skilledworker, and are explained fully in the literature. See, e.g., Ausubel,et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons,Inc., NY, N.Y. (1987-2008), including all supplements; Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor,N.Y. (1989).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart. The specification also provides definitions of terms to helpinterpret the disclosure and claims of this application. In the event adefinition is not consistent with definitions elsewhere, the definitionset forth in this application will control.

RNAse H Enzymes and the RNAse H Domain

This application describes modified thermostable RNase H enzymecompositions for use in CataCleave™ reverse transcriptase PCR reactions.

RNase H hydrolyzes RNA in RNA-DNA hybrids. First identified in calfthymus, RNAse H has subsequently been described in a variety oforganisms. Indeed, RNase H activity appears to be ubiquitous ineukaryotes and bacteria. Although RNase Hs form a family of proteins ofvarying molecular weight and nucleolytic activity, substraterequirements appear to be similar for the various isotypes. For example,most RNase Hs studied to date function as endonucleases and requiredivalent cations (e.g., Mg^(2±), Mn²) to produce cleavage products with5′ phosphate and 3′ hydroxyl termini.

In prokaryotes, RNase H have been cloned and extensively characterized(see Crooke, et al., (1995) Biochem J, 312 (Pt 2), 599-608; Lima, etal., (1997) J Biol Chem, 272, 27513-27516; Lima, et al., (1997)Biochemistry, 36, 390-398; Lima, et al., (1997) J Biol Chem, 272,18191-18199; Lima, et al., (2007) Mol Pharmacol, 71, 83-91; Lima, etal., (2007) Mol Pharmacol, 71, 73-82; Lima, et al., (2003) J Biol Chem,278, 14906-14912; Lima, et al., (2003) J Biol Chem, 278, 49860-49867;Itaya, M., Proc. Natl. Acad. Sci. USA, 1990, 87, 8587-8591). Forexample, E. coli RNase HII is 213 amino acids in length whereas RNase HIis 155 amino acids long. E. coli RNase HII displays only 17% homologywith E. coli RNase HI. An RNase H cloned from S. typhimurium differedfrom E. coli RNase HI in only 11 positions and was 155 amino acids inlength (Itaya, M. and Kondo K., Nucleic Acids Res., 1991, 19,4443-4449).

Proteins that display RNase H activity have also been cloned andpurified from a number of viruses, other bacteria and yeast(Wintersberger, U. Pharmac. Ther., 1990, 48, 259-280). In many cases,proteins with RNase H activity appear to be fusion proteins in whichRNase H is fused to the amino or carboxy end of another enzyme, often aDNA or RNA polymerase. The RNase H domain has been consistently found tobe highly homologous to E. coli RNase HI, but because the other domainsvary substantially, the molecular weights and other characteristics ofthe fusion proteins vary widely.

In higher eukaryotes two classes of RNase H have been defined based ondifferences in molecular weight, effects of divalent cations,sensitivity to sulfhydryl agents and immunological cross-reactivity(Busen et al., Eur. J. Biochem., 1977, 74, 203-208). RNase HI enzymesare reported to have molecular weights in the 68-90 kDa range, beactivated by either Mn²⁺ or Mg²⁺ and be insensitive to sulfhydrylagents. In contrast, RNase H II enzymes have been reported to havemolecular weights ranging from 31-45 kDa, to require Mg²⁺ to be highlysensitive to sulfhydryl agents and to be inhibited by Mn²⁺ (Busen, W.,and Hausen, P., Eur. J. Biochem., 1975, 52, 179-190; Kane, C. M.,Biochemistry, 1988, 27, 3187-3196; Busen, W., J. Biol. Chem., 1982, 257,7106-7108)

A detailed comparison of RNAses from different species is reported inOhtani N, Haruki M, Morikawa M, Kanaya S. J Biosci Bioeng. 1999;88(1):12-9.

An enzyme with RNase HII characteristics has also been purified to nearhomogeneity from human placenta (Frank et al., Nucleic Acids Res., 1994,22, 5247-5254). This protein has a molecular weight of approximately 33kDa and is active in a pH range of 6.5-10, with a pH optimum of 8.5-9.The enzyme requires Mg²⁺ and is inhibited by Mn²⁺ and n-ethyl maleimide.The products of cleavage reactions have 3′ hydroxyl and 5′ phosphatetermini.

Examples of RNase H enzymes, which may be employed in the embodiments,also include, but are not limited to, thermostable RNAse H enzymesisolated from thermophilic organisms such as Pyrococcus furiosus RNaseHII, Pyrococcus horikoshi RNase HII, Thermococcus litoralis RNase HI,Therms thermophilus RNase HI. Other RNAse H enzymes that may be employedin the embodiments are described in, for example, U.S. Pat. No.7,422,888 to Uemori or the published U.S. Patent Application No.2009/0325169 to Walder, the contents of which are incorporated herein byreference.

In one embodiment, an RNAse H enzyme is a thermostable RNAse H with 40%,50%, 60%, 70%, 80%, 90%, 95% or 99% homology with the amino acidsequence of Pfu RNase HII (SEQ ID NO: 1), shown below.

MKIGGIDEAG RGPAIGPLVV ATVVVDEKNI EKLRNIGVKD SKQLTPHERK NLFSQITSIA 60DDYKIVIVSP EEIDNRSGTM NELEVEKFAL ALNSLQIKPA LIYADAADVD ANRFASLIER 120RLNYKAKIIA EHKADAKYPV VSAASILAKV VRDEEIEKLK KQYGDFGSGY PSDPKTKKWL 180EEYYKKHNSF PPIVRRTWET VRKIEESIKA KKSQLTLDKF FKKP 224

The homology can be determined using, for example, a computer programDNASIS-Mac (Takara Shuzo), a computer algorithm FASTA (version 3.0;Pearson, W. R. et al., Pro. Natl. Acad. Sci., 85:2444-2448, 1988) or acomputer algorithm BLAST (version 2.0, Altschul et al., Nucleic AcidsRes. 25:3389-3402, 1997).

In another embodiment, an RNAse H enzyme is a thermostable RNAse H withat least one or more homology regions 1-4 corresponding to positions5-20, 33-44, 132-150, and 158-173 of SEQ ID NO: 1

HOMOLOGY REGION 1: GIDEAG RGPAIGPLVV (SEQ ID NO: 10; corresponding topositions 5-20 of SEQ ID NO: 1)

HOMOLOGY REGION 2: LRNIGVKD SKQL (SEQ ID NO: 11; corresponding topositions 33-44 of SEQ ID NO: 1)

HOMOLOGY REGION 3: HKADAKYPV VSAASILAKV (SEQ ID NO: 12; corresponding topositions 132-150 of SEQ ID NO: 1)

HOMOLOGY REGION 4: KLK KQYGDFGSGY PSD (SEQ ID NO: 13; corresponding topositions 158-173 of SEQ ID NO: 1)

In another embodiment, an RNAse H enzyme is a thermostable RNAse H withat least one of the homology regions having 50%, 60%. 70%, 80%, 90%sequence identity with a polypeptide sequence of SEQ ID NOs: 10, 11, 12,or 13.

The terms “sequence identity” as used herein refers to the extent thatsequences are identical or functionally or structurally similar on aamino acid to amino acid basis over a window of comparison. Thus, a“percentage of sequence identity”, for example, can be calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical amino acidoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

Modification of RNAse H

The term “modified RNase H,” as used herein, can be an RNase H reverselycoupled to or reversely bound to an inhibiting factor that causes theloss of the endonuclease activity of the RNase H. Release or decouplingof the inhibiting factor from the RNase H restores at least partial orfull activity of the endonuclease activity of the RNase H. About 30-100%of its activity of an intact RNase H may be sufficient. The inhibitingfactor may be a ligand or a chemical modification. The ligand can be anantibody, an aptamer, a receptor, a cofactor, or a chelating agent. Theligand can bind to the active site of the RNAse H enzyme therebyinhibiting enzymatic activity or it can bind to a site remote from theRNAse's active site. In some embodiment, the ligand may induce aconformational change. The chemical modification can be a crosslinking(for example, by formaldehyde) or acylation. The release or decouplingof the inhibiting factor from the RNase HII may be accomplished byheating a sample or a mixture containing the coupled RNase HII(inactive) to a temperature of about 65° C. to about 95° C. or higher,and/or lowering the pH of the mixture or sample to about 7.0 or lower.

The term “inactivated RNase H” or “inactive RNase H,” as used herein,refers to an RNase H, which lost its endonuclease activity by more thanabout 75% or by more than about 85% or by more than about 95% ascompared to unmodified RNase H (considered as 100%) determined at 50° C.under otherwise identical experimental conditions.

The term “activated RNase H” or “active RNase H,” as used herein, refersto an RNase HII, which has been modified as described above, recoversits endonuclease activity by more than about 5% or about 10% or about15% or about 20% or about 25% or about 30% or more as compared tounmodified RNase H (considered as 100%) determined at 50° C. underotherwise identical experimental conditions.

As used herein, an “inducible” RNAse H activity refers to the hereindescribed modified RNAse H that has an endonuclease catalytic activitythat can be regulated by association with a ligand. Under permissiveconditions, the RNAse H endonuclease catalytic activity is activatedwhereas at non-permissive conditions, this catalytic activity isinhibited. In some embodiments, the catalytic activity of a modifiedRNAse H can be inhibited at temperature conducive for reversetranscription, i.e. about 42° C., and activated at more elevatedtemperatures found in PCR reactions, i.e. about 65° C. to 95° C. Amodified RNAse H with these characteristics is said to be “heatinducible.”

In other embodiments, the catalytic activity of a modified RNAse H canbe regulated by changing the pH of a solution containing the enzyme.

As used herein, a “hot start” enzyme composition refers to compositionshaving an enzymatic activity that is inhibited at non-permissivetemperatures, i.e. from about 25° C. to about 45° C. and activated attemperatures compatible with a PCR reaction, e.g. about 55° C. to about95° C. In certain embodiment, a “hot start” enzyme composition may havea ‘hot start’ RNAse H and/or a ‘hot start’ thermostable DNA polymerasethat are known in the art.

Crosslinking of RNAse H enzymes can be performed using, for example,formaldehyde. In one embodiment, a thermostable RNase HII is subjectedto controlled and limited crosslinking using formaldehyde. By heating anamplification reaction composition, which comprises the modified RNaseHII in an inactive state, to a temperature of about 95° C. or higher foran extended time, for example about 15 minutes, the crosslinking isreversed and the RNase HII activity is restored.

In general, the lower the degree of crosslinking, the higher theendonuclease activity of the enzyme is after reversal of crosslinking.The degree of crosslinking may be controlled by varying theconcentration of formaldehyde and the duration of crosslinking reaction.For example, about 0.2% (w/v), about 0.4% (w/v), about 0.6% (w/v), orabout 0.8% (w/v) of formaldehyde may be used to crosslink an RNase Henzyme. About 10 minutes of crosslinking reaction using 0.6%formaldehyde may be sufficient to inactivate RNase HII from Pyrococcusfuriosus.

The crosslinked RNase HII does not show any measurable endonucleaseactivity at about 37° C. In some cases, a measurable partialreactivation of the crosslinked RNase HII may occur at a temperature ofaround 50° C., which is lower than the PCR denaturation temperature. Toavoid such unintended reactivation of the enzyme, it may be required tostore or keep the modified RNase HII at a temperature lower than 50° C.until its reactivation.

In general, PCR requires heating the amplification composition at eachcycle to about 95° C. to denature the double stranded target sequencewhich will also release the inactivating factor from the RNase H,partially or fully restoring the activity of the enzyme.

RNase H may also be modified by subjecting the enzyme to acylation oflysine residues using an acylating agent, for example, a dicarboxylicacid. Acylation of RNase H may be performed by adding cis-aconiticanhydride to a solution of RNase H in an acylation buffer and incubatingthe resulting mixture at about 1-20° C. for 5-30 hours. In oneembodiment, the acylation may be conducted at around 3-8° C. for 18-24hours. The type of the acylation buffer is not particularly limited. Inan embodiment, the acylation buffer has a pH of between about 7.5 toabout 9.0.

The activity of acylated RNase H can be restored by lowering the pH ofthe amplification composition to about 7.0 or less. For example, whenTris buffer is used as a buffering agent, the composition may be heatedto about 95° C., resulting in the lowering of pH from about 8.7 (at 25°C.) to about 6.5 (at 95° C.).

The duration of the heating step in the amplification reactioncomposition may vary depending on the modified RNase H, the buffer usedin the PCR, and the like. However, in general, heating the amplificationcomposition to 95° C. for about 30 seconds-4 minutes is sufficient torestore RNase H activity. In one embodiment, using a commerciallyavailable buffer such as Invitrogen AgPath™ buffer, full activity ofPyrococcus furiosus RNase HII is restored after about 2 minutes ofheating.

RNase H activity may be determined using methods that are well known inthe art. For example, according to a first method, the unit activity isdefined in terms of the acid-solubilization of a certain number of molesof radiolabeled polyadenylic acid in the presence of equimolarpolythymidylic acid under defined assay conditions (see EpicentreHybridase thermostable RNase HI). In the second method, unit activity isdefined in terms of a specific increase in the relative fluorescenceintensity of a reaction containing equimolar amounts of the probe and acomplementary template DNA under defined assay conditions. This secondmethod is explained in more detail in the working Examples.

Methods of using a modified RNAse H according to the invention arehereby disclosed in the context of an exemplary embodiment of detectingan RNA sequence in a test sample using Catacleave™ reverse transcriptasePCR detection.

Nucleic Acid Template Preparation

In some embodiments, the sample comprises a purified nucleic acidtemplate (e.g., mRNA, rRNA, and mixtures thereof). Procedures for theextraction and purification of RNA from samples are well known in theart. For example, RNA can be isolated from cells using the TRIzol™reagent (Invitrogen) extraction method. RNA quantity and quality is thendetermined using, for example, a Nanodrop™ spectrophotometer and anAgilent 2100 bioanalyzer.

In other embodiments, the sample is a cell lysate that is produced bylysing cells using a lysis buffer having a pH of about 6 to about 9, azwitterionic detergent at a concentration of about 0.125% to about 2%,an azide at a concentration of about 0.3 to about 2.5 mg/ml and aprotease such as proteinase K (about 1 mg/ml). After incubation at 55°C. for 15 minutes, the proteinase K is inactivated at 95° C. for 10minutes to produce a “substantially protein free” lysate that iscompatible with high efficiency PCR or reverse transcription PCRanalysis.

In one embodiment, the 1× lysis reagent contains 12.5 mM Tris acetate orTris-HCl or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)(pH=7-8), 0.25% (w/v) CHAPS, 0.3125 mg/ml sodium azide and proteinase Kat 1 mg/ml.

The term “lysate” as used herein, refers to a liquid phase with lysedcell debris and nucleic acids.

As used herein, the term “substantially protein free” refers to a lysatewhere most proteins are inactivated by proteolytic cleavage by aprotease. Protease may include proteinase K. Addition of proteinase Kduring cell lysis rapidly inactivates nucleases that might otherwisedegrade the target nucleic acids. The “substantially protein free”lysate may be or may not be subjected to a treatment to removeinactivated proteins.

As used herein, the term “cells” can refer to prokaryotic or eukaryoticcells.

In one embodiment, the term “cells” can refer to microorganisms such asbacteria including, but not limited to gram positive bacteria, gramnegative bacteria, acid-fast bacteria and the like. In certainembodiments, the “cells” to be tested may be collected using swabsampling of surfaces. In other embodiments, the “cells” can refer topathogenic organisms.

In other embodiments, the sample comprises a viral nucleic acid, forexample, a retroviral nucleic acid. In certain embodiment, a sample maycontain a lentiviral nucleic acid such as HIV-1 or HIV-2.

As used herein, “zwitterionic detergent” refers to detergents exhibitingzwitterionic character (e.g., does not possess a net charge, lacksconductivity and electrophoretic mobility, does not bind ion-exchangeresins, breaks protein-protein interactions), including, but not limitedto, CHAPS, CHAPSO and betaine derivatives, e.g. preferably sulfobetainessold under the brand names Zwittergent® (Calbiochem, San Diego, Calif.)and Anzergent® (Anatrace, Inc. Maumee, Ohio).

In one embodiment, the zwitterionic detergent is CHAPS (CAS Number:75621-03-3; available from SIGMA-ALDRICH product no. C3023-1G), anabbreviation for3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (described infurther detail in U.S. Pat. No. 4,372,888) having the structure:

In a further embodiment, CHAPS is present at a concentration of about0.125% to about 2% weight/volume (w/v) of the total composition. In afurther embodiment, CHAPS is present at a concentration of about 0.25%to about 1% w/v of the total composition. In yet another embodiment,CHAPS is present at a concentration of about 0.4% to about 0.7% w/v ofthe total composition.

In other embodiments, the lysis buffer may include other non-ionicdetergents such as Nonidet, Tween or Triton X-100.

As used herein, the term “buffer” refers to a composition that caneffectively maintain the pH value between 6 and 9, with a pK_(a) at 25°C. of about 6 to about 9. The buffer described herein is generally aphysiologically compatible buffer that is compatible with the functionof enzyme activities and enables biological macromolecules to retaintheir normal physiological and biochemical functions.

Examples of buffers include, but are not limited to, HEPES((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(3-(N-morpholino)-propanesulfonic acid),N-tris(hydroxymethyl)methylglycine acid (Tricine),tris(hydroxymethyl)methylamine acid (Tris),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) and acetate orphosphate containing buffers (K₂HPO₄, KH₂PO₄, Na₂HPO₄, NaH₂PO₄) and thelike.

The term “azide” as used herein is represented by the formula —N₃. Inone embodiment, the azide is sodium azide NaN₃ (CAS number 26628-22-8;available from SIGMA-ALDRICH Product number: S2002-25G) that acts as ageneral bacterioside.

The term “protease,” as used herein, is an enzyme that hydrolysespeptide bonds (has protease activity). Proteases are also called, e.g.,peptidases, proteinases, peptide hydrolases, or proteolytic enzymes. Theproteases for use according to the invention can be of the endo-typethat act internally in polypeptide chains (endopeptidases). In oneembodiment, the protease can be the serine protease, proteinase K (EC3.4.21.64; available from Roche Applied Sciences, recombinant proteinaseK 50 U/ml (from Pichia pastoris) Cat. No. 03 115 887 001).

Proteinase K is used to digest protein and remove contamination frompreparations of nucleic acid. Addition of proteinase K to nucleic acidpreparations rapidly inactivates nucleases that might otherwise degradethe DNA or RNA during purification. It is highly-suited to thisapplication since the enzyme is active in the presence of chemicals thatdenature proteins and it can be inactivated at temperatures of about 95°C. for about 10 minutes.

In one embodiment, lysis of gram positive and gram negative bacteria,such as Listeria, Salmonella, and E. Coli also requires the lysisreagent include proteinase K (1 mg/ml). Protein in the cell lysate isdigested by proteinase K for 15 minutes at 55° C. followed byinactivation of the proteinase K at 95° C. for 10 minutes. Aftercooling, the substantially protein free lysate is compatible with highefficiency PCR amplification.

In addition to or in lieu of proteinase K, the lysis reagent cancomprise a serine protease such as trypsin, chymotrypsin, elastase,subtilisin, streptogrisin, thermitase, aqualysin, plasmin, cucumisin, orcarboxypeptidase A, D, C, or Y. In addition to a serine protease, thelysis solution can comprise a cysteine protease such as papain, calpain,or clostripain; an acid protease such as pepsin, chymosin, or cathepsin;or a metalloprotease such as pronase, thermolysin, collagenase, dispase,an aminopeptidase or carboxypeptidase A, B, E/H, M, T, or U. ProteinaseK is stable over a wide pH range (pH 4.0-10.0) and is stable in bufferswith zwitterionic detergents.

Reverse Transcriptase-PCR Amplification

The reverse transcriptase-PCR procedure can be set up as either anend-point or real-time assay. cDNA amplification requires essentiallytwo separate molecular syntheses: (i) the synthesis of cDNA from an RNAtemplate; and (ii) the replication of the newly synthesized cDNA throughPCR amplification. To attempt to address the technical problems oftenassociated with reverse transcriptase-PCR, a number of protocols havebeen developed taking into account the three basic steps of theprocedure: (a) the denaturation of RNA and the hybridization of reverseprimer; (b) the synthesis of cDNA; and (c) PCR amplification. In the socalled “uncoupled” reverse transcriptase-PCR procedure (e.g., two stepreverse transcriptase-PCR), reverse transcription is performed as anindependent step using the optimal buffer condition for reversetranscriptase activity. Following cDNA synthesis, the reaction isdiluted to decrease MgCl₂, and deoxyribonucleoside triphosphate (dNTP)concentrations to conditions optimal for Taq DNA Polymerase activity,and PCR is carried out according to standard conditions (see U.S. Pat.Nos. 4,683,195 and 4,683,202). By contrast, “coupled” reversetranscriptase PCR methods use a common buffer for reverse transcriptaseand Taq DNA Polymerase activities. In one version, the annealing ofreverse primer is a separate step preceding the addition of enzymes,which are then added to the single reaction vessel. In another version,the reverse transcriptase activity is a component of the thermostableTth DNA polymerase. Annealing and cDNA synthesis are performed in thepresence of Mn²⁺ then PCR is carried out in the presence of Mg²⁺ afterthe removal of Mn²⁺ by a chelating agent. Finally, the “continuous”method (e.g., one step reverse transcriptase-PCR) integrates the threereverse transcriptase-PCR steps into a single continuous reaction thatavoids the opening of the reaction tube for component or enzymeaddition. Continuous reverse transcriptase-PCR has been described as asingle enzyme system using the reverse transcriptase activity ofthermostable Taq DNA Polymerase and Tth polymerase and as a two enzymesystem using AMV reverse transcriptase and Taq DNA Polymerase whereinthe initial 65° C. RNA denaturation step is omitted.

To maintain the highest sensitivity it is important that the RNA not bedegraded prior to cDNA synthesis. As noted above, the presence of RNaseH in one step reverse transcription PCR protocols can cause unwanteddegradation of the RNA:DNA primer hybrid before it can serve as asubstrate for reverse transcriptase. The modified RNase H describedherein resolves this issue by inactivating RNAse H endonucleasecatalytic activity at temperatures required for reverse transcription,i.e. about 45-55° C. For example, a hot start RNAse H activity, as usedherein, can be an RNAse H with a reversible chemical modificationproduced after reaction of the RNAse H with cis-aconitic anhydride underalkaline conditions. When the modified enzyme is used in a reaction witha Tris based buffer and the temperature is raised to 95° C. the pH ofthe solution drops and RNase H activity is restored. This method allowsfor the inclusion of RNase H in the reaction mixture prior to theinitiation of reverse transcription.

The first step in real-time, reverse-transcription PCR is to generatethe complementary DNA strand using one of the template specific DNAprimers. In traditional PCR reactions this product is denatured, thesecond template specific primer binds to the cDNA, and is extended toform duplex DNA. This product is then amplified in subsequent rounds ofPCR amplification.

The term “polymerase chain reaction” (PCR) refers to a method foramplification well known in the art for increasing the concentration ofa segment of a target polynucleotide in a sample, where the sample canbe a single polynucleotide species, or multiple polynucleotides.Generally, the PCR process consists of introducing a molar excess of twoor more extendable oligonucleotide primers to a 10-100 μl reactionmixture comprising a sample, a buffering agent, a thermostable DNApolymerase, target nucleic acid sequence(s), dNTPs (each of the fourdeoxynucleotides dATP, dCTP, dGTP, and dTTP), and primers that arecomplementary to opposite strands of the double stranded target sequencein the sample. The reaction mixture is subjected to a program of thermalcycling in the presence of a DNA polymerase, resulting in theamplification of the desired target sequence flanked by the DNA primers.One PCR reaction may consist of about 5 to about 100 “cycles” ofdenaturation and synthesis of a polynucleotide molecule.

The technique of PCR is described in numerous publications, including,PCR: A Practical Approach, M. J. McPherson, et al., IRL Press (1991),PCR Protocols: A Guide to Methods and Applications, by Innis, et al.,Academic Press (1990), and PCR Technology: Principals and Applicationsfor DNA Amplification, H. A. Erlich, Stockton Press (1989). PCR is alsodescribed in many U.S. patents, including U.S. Pat. Nos. 4,683,195;4,683,202; 4,800,159; 4,965,188; 4,889,818; 5,075,216; 5,079,352;5,104,792; 5,023,171; 5,091,310; and 5,066,584, each of which is hereinincorporated by reference.

The term “nucleotide,” as used herein, refers to a compound comprising anucleotide base linked to the C-1′ carbon of a sugar, such as ribose,arabinose, xylose, and pyranose, and sugar analogs thereof. The termnucleotide also encompasses nucleotide analogs. The sugar may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those riboses in which one or more of the carbon atoms,for example the 2′-carbon atom, is substituted with one or more of thesame or different Cl, F, —R, —OR, —NR2 or halogen groups, where each Ris independently H, C1-C6 alkyl or C5-C14 aryl. Exemplary ribosesinclude, but are not limited to, 2′-(C1-C6)alkoxyribose,2′-(C5-C14)aryloxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose,2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C1-C6)alkylribose,2′-deoxy-3′-(C1-C6)alkoxyribose and 2′-deoxy-3′-(C5-C14)aryloxyribose,ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-α-anomeric nucleotides, 1′-α-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(see, e.g., PCT published application nos. WO 98/22489, WO 98/39352, andWO 99/14226; and U.S. Pat. Nos. 6,268,490 and 6,794,499).

As used herein, the term “nucleic acid” refers to an oligonucleotide orpolynucleotide, wherein said oligonucleotide or polynucleotide may bemodified or may comprise modified bases. Oligonucleotides aresingle-stranded polymers of nucleotides comprising from 2 to 60nucleotides. Polynucleotides are polymers of nucleotides comprising twoor more nucleotides. Polynucleotides may be either double-stranded DNAs,including annealed oligonucleotides wherein the second strand is anoligonucleotide with the reverse complement sequence of the firstoligonucleotide, single-stranded nucleic acid polymers comprisingdeoxythymidine, single-stranded RNAs, double stranded RNAs or RNA/DNAheteroduplexes. Nucleic acids include, but are not limited to, genomicDNA, RNA, cDNA, hnRNA, snRNA, mRNA, rRNA, tRNA, miRNA, siRNA, fragmentednucleic acid, nucleic acid obtained from subcellular organelles such asmitochondria or chloroplasts, and nucleic acid obtained frommicroorganisms or DNA or RNA viruses that may be present on or in abiological sample. Nucleic acids may be composed of a single type ofsugar moiety, e.g., as in the case of RNA and DNA, or mixtures ofdifferent sugar moieties, e.g., as in the case of RNA/DNA chimeras. Inthe specification, the nucleotides “A,” “C,” and “G” may be either adeoxyribonucleotide or ribonucleotide, and ribonucleotide A,ribonucleotide C, and ribonucleotide G are indicated by “rA,” “rC,” and“rG,” respectively in the sequences of oligonucleotides.

A “target DNA” or “target RNA”” or “target nucleic acid,” or “targetnucleic acid sequence” refers to a nucleic acid that is targeted for DNAamplification. A target nucleic acid sequence serves as a template foramplification in a PCR reaction or reverse transcriptase-PCR reaction.Target nucleic acid sequences may include both naturally occurring andsynthetic molecules. Exemplary target nucleic acid sequences include,but are not limited to, genomic DNA or genomic RNA.

As used herein, the term “oligonucleotide” is used sometimesinterchangeably with “primer” or “polynucleotide.” The term “primer”refers to an oligonucleotide that acts as a point of initiation of DNAsynthesis in a PCR reaction. A primer is usually about 15 to about 35nucleotides in length and hybridizes to a region complementary to thetarget sequence.

Oligonucleotides may be synthesized and prepared by any suitable methods(such as chemical synthesis), which are known in the art.Oligonucleotides may also be conveniently available through commercialsources.

The terms “annealing” and “hybridization” are used interchangeably andmean the base-pairing interaction of one nucleic acid with anothernucleic acid that results in formation of a duplex, triplex, or otherhigher-ordered structure. In certain embodiments, the primaryinteraction is base specific, e.g., A/T and G/C, by Watson/Crick andHoogsteen-type hydrogen bonding. In certain embodiments, base-stackingand hydrophobic interactions may also contribute to duplex stability.

A “buffering agent” or “buffer” is a compound added to an amplificationreaction which modifies the stability, activity, and/or longevity of oneor more components of the amplification reaction by regulating the pH ofthe amplification reaction. Certain buffering agents are well known inthe art and include, but are not limited to, Tris, Tricine, MOPS(3-(N-morpholino)propanesulfonic acid), and HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

The term “sample” refers to any substance containing nucleic acidmaterial.

An additive is a compound added to a composition which modifies thestability, activity, and/or longevity of one or more components of thecomposition. In certain embodiments, the composition is an amplificationreaction composition. In certain embodiments, an additive inactivatescontaminant enzymes, stabilizes protein folding, and/or decreasesaggregation. Exemplary additives that may be included in anamplification reaction include, but are not limited to, betaine,formamide, KCl, CaCl2, MgOAc, MgCl2, NaCl, NH4OAc, NaI, Na(CO3)2, LiCl,MnOAc, NMP, trehalose, demethylsulfoxide (“DMSO”), glycerol, ethyleneglycol, dithiothreitol (“DTT”), pyrophosphatase (including, but notlimited to Thermoplasma acidophilum inorganic pyrophosphatase (“TAP”)),bovine serum albumin (“BSA”), propylene glycol, glycinamide, CHES,Percoll™, aurintricarboxylic acid, Tween 20, Tween 21, Tween 40, Tween60, Tween 85, Brij 30, NP-40, Triton X-100, CHAPS, CHAPSO, Mackernium,LDAO (N-dodecyl-N,N-dimethylamine-N-oxide), Zwittergent 3-10,Xwittergent 3-14, Xwittergent SB 3-16, Empigen, NDSB-20, T4G32, E. ColiSSB, RecA, nicking endonucleases, 7-deazaG, dUTP, and UNG, anionicdetergents, cationic detergents, non-ionic detergents, zwittergent,sterol, osmolytes, cations, and any other chemical, protein, or cofactorthat may alter the efficiency of amplification. In certain embodiments,two or more additives are included in an amplification reaction.

As used herein, “DNA polymerase activity” refers to an enzymaticactivity that catalyzes the polymerization of deoxyribonucleotides.Generally, the enzyme will initiate synthesis at the 3 ‘-end of theprimer annealed to a nucleic acid template sequence, and will proceedtoward the 5’ end of the template strand. Known DNA polymerases include,for example, Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al.,1991, Gene, 108:1), E. coli DNA polymerase I (Lecomte and Doubleday,1983, Nucleic Acids Res. 11:7505), T7 DNA polymerase (Nordstrom et al.,1981, J. Biol. Chem. 256:3112), Thermus thermophilus (Tth) DNApolymerase (Myers and Gelfand 1991, Biochemistry 30:7661), Bacillusstearothermophilus DNA polymerase (Stenesh and McGowan, 1977, BiochimBiophys Acta 475:32), Thermococcus litoralis (TIi) DNA polymerase (alsoreferred to as Vent DNA polymerase, Cariello et al., 1991, Nucleic AcidsRes, 19: 4193), 9° Nm DNA polymerase (discontinued product from NewEngland Biolabs), Thermotoga maritima (Tma) DNA polymerase (Diaz andSabino, 1998 Braz J. Med. Res, 31:1239), Thermus aquaticus (Taq) DNApolymerase (Chien et al., 1976, J. Bacteoriol, 127: 1550), Pyrococcuskodakaraensis KOD DNA polymerase (Takagi et al., 1997, Appl. Environ.Microbiol. 63:4504), JDF-3 DNA polymerase (Patent application WO0132887), and Pyrococcus GB-D (PGB-D) DNA polymerase (Juncosa-Ginesta etal., 1994, Biotechniques, 16:820). The polymerase activity of any of theabove enzymes can be determined by means well known in the art. One unitof DNA polymerase activity, according to the subject invention, isdefined as the amount of enzyme which catalyzes the incorporation of 10nmoles of total dNTPs into polymeric form in 30 minutes at optimaltemperature (e.g., 72° C. for Pfu DNA polymerase).

The term “reverse transcriptase activity” and “reverse transcription”refers to the enzymatic activity of a class of polymerases characterizedas RNA-dependent DNA polymerases that can synthesize a DNA strand (i.e.,complementary DNA, cDNA) utilizing an RNA strand as a template.

“Reverse transcriptase-PCR” is a PCR reaction that uses RNA template anda reverse transcriptase, or an enzyme having reverse transcriptaseactivity, to first generate a single stranded DNA molecule prior to themultiple cycles of DNA-dependent DNA polymerase primer elongation.Multiplex PCR refers to PCR reactions that produce more than oneamplified product in a single reaction, typically by the inclusion ofmore than two primers in a single reaction.

Exemplary reverse transcriptases include, but are not limited to, theMoloney murine leukemia virus (M-MLV) RT as described in U.S. Pat. No.4,943,531, a mutant form of M-MLV-RT lacking RNase H activity asdescribed in U.S. Pat. No. 5,405,776, bovine leukemia virus (BLV) RT,Rous sarcoma virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT andreverse transcriptases disclosed in U.S. Pat. No. 7,883,871.

For PCR amplifications, the enzymes used in the invention are preferablythermostable. The term “thermostable” refers to an enzyme which isstable to heat, is heat resistant, and functions at high temperatures,e.g., 50° C. to 90° C. The thermostable enzyme according to the presentinvention must satisfy a single criterion to be effective for theamplification reaction, i.e., the enzyme must not become irreversiblydenatured (inactivated) when subjected to the elevated temperatures forthe time necessary to effect denaturation of double-strandedpolynucleotides. By “irreversible denaturation” as used in thisconnection, is meant a process bringing a permanent and complete loss ofenzymatic activity. The heating conditions necessary for denaturationwill depend, e.g., on the buffer salt concentration and the length andnucleotide composition of the polynucleotides being denatured, buttypically range from about 85° C., for shorter polynucleotides, to about105° C. for a time depending mainly on the temperature and thepolynucleotide length, typically from about 0.25 minutes for shorterpolynucleotides, to about 4.0 minutes for longer pieces of DNA. Highertemperatures may be tolerated as the buffer salt concentration and/or GCcomposition of the polynucleotide is increased. Preferably, the enzymewill not become irreversibly denatured at about 90° C. to about 100° C.An enzyme that does not become irreversibly denatured, according to theinvention, retains at least about 10%, or at least about 25%, or atleast about 50% or more function or activity during the amplificationreaction.

In certain embodiments, one or more primers may be labeled. As usedherein, “label,” “detectable label,” or “marker,” or “detectablemarker,” which are interchangeably used in the specification, refers toany chemical moiety attached to a nucleotide, nucleotide polymer, ornucleic acid binding factor, wherein the attachment may be covalent ornon-covalent. Preferably, the label is detectable and renders thenucleotide or nucleotide polymer detectable to the practitioner of theinvention. Detectable labels include luminescent molecules,chemiluminescent molecules, fluorochromes, fluorescent quenching agents,colored molecules, radioisotopes or scintillants. Detectable labels alsoinclude any useful linker molecule (such as biotin, avidin,streptavidin, HRP, protein A, protein G, antibodies or fragmentsthereof, Grb2, polyhistidine, Ni²⁺, FLAG tags, myc tags), heavy metals,enzymes (examples include alkaline phosphatase, peroxidase andluciferase), electron donors/acceptors, acridinium esters, dyes andcalorimetric substrates. It is also envisioned that a change in mass maybe considered a detectable label, as is the case of surface plasmonresonance detection. The skilled artisan would readily recognize usefuldetectable labels that are not mentioned above, which may be employed inthe operation of the present invention.

One step reverse transcriptase-PCR provides several advantages overuncoupled reverse transcriptase-PCR. One step reverse transcriptase-PCRrequires less handling of the reaction mixture reagents and nucleic acidproducts than uncoupled reverse transcriptase-PCR (e.g., opening of thereaction tube for component or enzyme addition in between the tworeaction steps), and is therefore less labor intensive, reducing therequired number of person hours. One step reverse transcriptase-PCR alsorequires less sample, and reduces the risk of contamination. Thesensitivity and specificity of one-step reverse transcriptase-PCR hasproven well suited for studying expression levels of one to severalgenes in a given sample or the detection of pathogen RNA. Typically,this procedure has been limited to the use of gene-specific primers toinitiate cDNA synthesis.

The ability to measure the kinetics of a PCR reaction by real-timedetection in combination with these reverse transcriptase-PCR techniqueshas enabled accurate and precise determination of RNA copy number withhigh sensitivity. This has become possible by detecting the reversetranscriptase-PCR product through fluorescence monitoring andmeasurement of PCR product during the amplification process byfluorescent dual-labeled hybridization probe technologies, such as the5′ fluorogenic nuclease assay (“Taq-Man”) or endonuclease assay(“CataCleave”), discussed below.

Real-Time PCR of a Salmonella Target Nucleic Acid Sequence Using aCataCleave Probe

Post-amplification amplicon detection is both laborious and timeconsuming. Real-time methods have been developed to monitoramplification during the PCR process. These methods typically employfluorescently labeled probes that bind to the newly synthesized DNA ordyes whose fluorescence emission is increased when intercalated intodouble stranded DNA.

The probes are generally designed so that donor emission is quenched inthe absence of target by fluorescence resonance energy transfer (FRET)between two chromophores. The donor chromophore, in its excited state,may transfer energy to an acceptor chromophore when the pair is in closeproximity. This transfer is always non-radiative and occurs throughdipole-dipole coupling. Any process that sufficiently increases thedistance between the chromophores will decrease FRET efficiency suchthat the donor chromophore emission can be detected radiatively. Commondonor chromophores include FAM, TAMRA, VIC, JOE, Cy3, Cy5, and TexasRed. Acceptor chromophores are chosen so that their excitation spectraoverlap with the emission spectrum of the donor. An example of such apair is FAM-TAMRA. There are also non fluorescent acceptors that willquench a wide range of donors. Other examples of appropriatedonor-acceptor FRET pairs will be known to those skilled in the art.

Common examples of FRET probes that can be used for real-time detectionof PCR include molecular beacons (e.g., U.S. Pat. No. 5,925,517 TaqManprobes (e.g., U.S. Pat. Nos. 5,210,015 and 5,487,972), and CataCleaveprobes (e.g., U.S. Pat. No. 5,763,181). The molecular beacon is a singlestranded oligonucleotide designed so that in the unbound state the probeforms a secondary structure where the donor and acceptor chromophoresare in close proximity and donor emission is reduced. At the properreaction temperature the beacon unfolds and specifically binds to theamplicon. Once unfolded the distance between the donor and acceptorchromophores increases such that FRET is reversed and donor emission canbe monitored using specialized instrumentation. TaqMan and CataCleavetechnologies differ from the molecular beacon in that the FRET probesemployed are cleaved such that the donor and acceptor chromophoresbecome sufficiently separated to reverse FRET.

TaqMan technology employs a single stranded oligonucleotide probe thatis labeled at the 5′ end with a donor chromophore and at the 3′ end withan acceptor chromophore. The DNA polymerase used for amplification mustcontain a 5′->3′ exonuclease activity. The TaqMan probe binds to onestrand of the amplicon at the same time that the primer binds. As theDNA polymerase extends the primer the polymerase will eventuallyencounter the bound TaqMan probe. At this time the exonuclease activityof the polymerase will sequentially degrade the TaqMan probe starting atthe 5′ end. As the probe is digested the mononucleotides comprising theprobe are released into the reaction buffer. The donor diffuses awayfrom the acceptor and FRET is reversed. Emission from the donor ismonitored to identify probe cleavage. Because of the way TaqMan works aspecific amplicon can be detected only once for every cycle of PCR.Extension of the primer through the TaqMan target site generates adouble stranded product that prevents further binding of TaqMan probesuntil the amplicon is denatured in the next PCR cycle.

U.S. Pat. No. 5,763,181, the content of which is incorporated herein byreference, describes another real-time detection method (referred to as“CataCleave” herein). CataCleave technology differs from TaqMan in thatcleavage of the probe is accomplished by a second enzyme that does nothave polymerase activity. The CataCleave probe has a sequence within themolecule which is a target of an endonuclease, such as, for example arestriction enzyme or RNAase. In one example, the CataCleave probe has achimeric structure where the 5′ and 3′ ends of the probe are constructedof DNA and the cleavage site contains RNA. The DNA sequence portions ofthe probe are labeled with a FRET pair either at the ends or internally.The PCR reaction includes an RNase H enzyme that will specificallycleave the RNA sequence portion of a RNA-DNA duplex (see FIG. 2). Aftercleavage, the two halves of the probe dissociate from the targetamplicon at the reaction temperature and diffuse into the reactionbuffer. As the donor and acceptors separate FRET is reversed in the sameway as the TaqMan probe and donor emission can be monitored. Cleavageand dissociation regenerates a site for further CataCleave binding. Inthis way it is possible for a single amplicon to serve as a target ormultiple rounds of probe cleavage until the primer is extended throughthe CataCleave probe binding site.

Labeling of a CataCleave Probe

The term “probe” comprises a polynucleotide having a specific portiondesigned to hybridize in a sequence-specific manner with a complementaryregion of a specific nucleic acid sequence, e.g., a target nucleic acidsequence. In one embodiment, the oligonucleotide probe is in the rangeof about 15 to about 60 nucleotides in length. In another embodiments,the oligonucleotide probe is in the range of about 18 to about 30nucleotides in length. The precise sequence and length of anoligonucleotide probe depends in part on the nature of the targetpolynucleotide to which it binds. The binding location and length may bevaried to achieve appropriate annealing and melting properties for aparticular embodiment. Guidance for making such design choices can befound in many of the references describing Taq-man assays or CataCleave,described in U.S. Pat. Nos. 5,763,181, 6,787,304, and 7,112,422, thecontents of which contents are incorporated herein by reference in theirentirety.

The probe may be labeled with a “label” or “detectable label” asdiscussed above. In an embodiment, the label is a fluorochrome compoundthat is attached to the probe by covalent or non-covalent means.

As used herein, “fluorochrome” refers to a fluorescent compound thatemits light upon excitation by light of a shorter wavelength than thelight that is emitted. The term “fluorescent donor” or “fluorescencedonor” refers to a fluorochrome that emits light that is measured in theassays described in the present invention. More specifically, afluorescent donor provides energy (The transfer is non radiative, thatis why I removed the word “light” that is absorbed by a fluorescenceacceptor. The term “fluorescent acceptor” or “fluorescence acceptor”refers to either a second fluorochrome or a quenching molecule thatabsorbs energy emitted from the fluorescence donor. The secondfluorochrome absorbs the energy that is emitted from the fluorescencedonor and emits light of longer wavelength than the light emitted by thefluorescence donor. The quenching molecule absorbs energy emitted by thefluorescence donor.

Any luminescent molecule, preferably a fluorochrome and/or fluorescentquencher may be used in the practice of this invention, including, forexample, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633,Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680,7-diethylaminocoumarin-3-carboxylic acid, Fluorescein, Oregon Green 488,Oregon Green 514, Tetramethylrhodamine, Rhodamine X, Texas Red dye, QSY7, QSY33, Dabcyl, BODIPY FL, BODIPY 630/650, BODIPY 6501665, BODIPYTMR-X, BODIPY TR-X, Dialkylaminocoumarin, Cy5.5, Cy5, Cy3.5, Cy3,DTPA(Eu3+)-AMCA and TTHA(Eu3⁺)AMCA.

In one embodiment, the 3′ terminal nucleotide of the oligonucleotideprobe is blocked or rendered incapable of extension by a nucleic acidpolymerase. Such blocking is conveniently carried out by the attachmentof a reporter or quencher molecule to the terminal 3′ position of theprobe.

In another embodiment, reporter molecules are fluorescent organic dyesderivatized for attachment to the terminal 3′ or terminal 5′ ends of theprobe via a linking moiety. Preferably, quencher molecules are alsoorganic dyes, which may or may not be fluorescent, depending on theembodiment of the invention. For example, in a preferred embodiment ofthe invention, the quencher molecule is non-fluorescent. Generallywhether the quencher molecule is fluorescent or simply releases thetransferred energy from the reporter by non-radiative decay, theabsorption band of the quencher should substantially overlap thefluorescent emission band of the reporter molecule. Non-fluorescentquencher molecules that absorb energy from excited reporter molecules,but which do not release the energy radiatively, are referred to in theapplication as chromogenic molecules.

Exemplary reporter-quencher pairs may be selected from xanthene dyes,including fluoresceins, and rhodamine dyes. Many suitable forms of thesecompounds are widely available commercially with substituents on theirphenyl moieties which can be used as the site for bonding or as thebonding functionality for attachment to an oligonucleotide. Anothergroup of fluorescent compounds are the naphthylamines, having an aminogroup in the alpha or beta position. Included among such naphthylaminocompounds are 1-dimethylaminonaphthyl-5-sulfonate,1-anilino-8-naphthalene sulfonate and 2-p-touidinyl6-naphthalenesulfonate. Other dyes include 3-phenyl-7-isocyanatocoumarin, acridines,such as 9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes,pyrenes, and the like.

In one embodiment, reporter and quencher molecules are selected fromfluorescein and non-fluorescent quencher dyes.

There are many linking moieties and methodologies for attaching reporteror quencher molecules to the 5′ or 3′ termini of oligonucleotides, asexemplified by the following references: Eckstein, editor,Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford,1991); Zuckerman et al., Nucleic Acids Research, 15: 5305-5321 (1987)(3′ thiol group on oligonucleotide); Sharma et al., Nucleic AcidsResearch, 19: 3019 (1991) (3′ sulfhydryl); Giusti et al., PCR Methodsand Applications, 2: 223-227 (1993) and Fung et al., U.S. Pat. No.4,757,141 (5′ phosphoamino group via Aminolink. II available fromApplied Biosystems, Foster City, Calif.) Stabinsky, U.S. Pat. No.4,739,044 (3′ aminoalkylphosphoryl group); Agrawal et al., TetrahedronLetters, 31: 1543-1546 (1990) (attachment via phosphoramidate linkages);Sproat et al., Nucleic Acids Research, 15: 4837 (1987) (5′ mercaptogroup); Nelson et al., Nucleic Acids Research, 17: 7187-7194 (1989) (3′amino group); and the like.

Rhodamine and non-fluorescent quencher dyes are also convenientlyattached to the 3′ end of an oligonucleotide at the beginning of solidphase synthesis, e.g., Woo et al., U.S. Pat. No. 5,231,191; and Hobbs,Jr., U.S. Pat. No. 4,997,928.

Attachment of a CataCleave Probe to a Solid Support

In certain embodiments of the invention, the oligonucleotide probe canbe attached to a solid support. Different probes may be attached to thesolid support and may be used to simultaneously detect different targetsequences in a sample. Reporter molecules having different fluorescencewavelengths can be used on the different probes, thus enablinghybridization to the different probes to be separately detected.

Examples of preferred types of solid supports for immobilization of theoligonucleotide probe include polystyrene, avidin coated polystyrenebeads cellulose, nylon, acrylamide gel and activated dextran, controlledpore glass (CPG), glass plates and highly cross-linked polystyrene.These solid supports are preferred for hybridization and diagnosticstudies because of their chemical stability, ease of functionalizationand well defined surface area. Solid supports such as controlled poreglass (500 Å, 1000 Å) and non-swelling high cross-linked polystyrene(1000 Å) are particularly preferred in view of their compatibility witholigonucleotide synthesis.

The oligonucleotide probe may be present in a free form in a reactionsolution. Alternatively, the oligonucleotide probe may be attached tothe solid support in a variety of manners. For example, the probe may beattached to the solid support by attachment of the 3′ or 5′ terminalnucleotide of the probe to the solid support. However, the probe may beattached to the solid support by a linker which serves to distance theprobe from the solid support. The linker is most preferably at least 30atoms in length, more preferably at least 50 atoms in length.

Hybridization of a probe immobilized to a solid support generallyrequires that the probe be separated from the solid support by at least30 atoms, more-preferably at least 50 atoms. In order to achieve thisseparation, the linker generally includes a spacer positioned betweenthe linker and the 3′ nucleoside. For oligonucleotide synthesis, thelinker arm is usually attached to the 3′-OH of the 3′ nucleoside by anester linkage which can be cleaved with basic reagents to free theoligonucleotide from the solid support.

A wide variety of linkers are known in the art which may be used toattach the oligonucleotide probe to the solid support. The linker may beformed of any compound which does not significantly interfere with thehybridization of the target sequence to the probe attached to the solidsupport. The linker may be formed of a homopolymeric oligonucleotidewhich can be readily added on to the linker by automated synthesis.Alternatively, polymers such as functionalized polyethylene glycol canbe used as the linker. Such polymers are preferred over homopolymericoligonucleotides because they do not significantly interfere with thehybridization of probe to the target oligonucleotide. Polyethyleneglycol is particularly preferred because it is commercially available,soluble in both organic and aqueous media, easy to functionalize, and iscompletely stable under oligonucleotide synthesis and post-synthesisconditions.

The linkages between the solid support, the linker and the probe arepreferably not cleaved during removal of base protecting groups underbasic conditions at high temperature. Examples of preferred linkagesinclude carbamate and amide linkages. Immobilization of a probe is wellknown in the art and one skilled in the art may determine theimmobilization conditions.

According to one embodiment of the method, the hybridization probe isimmobilized on a solid support. The oligonucleotide probe is contactedwith a sample of nucleic acids under conditions favorable forhybridization. In an unhybridized state, the fluorescent label isquenched by the quencher. On hybridization to the target, thefluorescent label is separated from the quencher resulting influorescence.

Immobilization of the hybridization probe to the solid support alsoenables the target sequence hybridized to the probe to be readilyisolated from the sample. In later steps, the isolated target sequencemay be separated from the solid support and processed (e.g., purified,amplified) according to methods well known in the art depending on theparticular needs of the researcher.

Real-Time Detection of Target Nucleic Acid Sequences Using a CataCleaveProbe

The labeled oligonucleotide probe may be used as a probe for thereal-time detection of a target nucleic acid sequence in a sample (seeFIGS. 1 and 2).

A CataCleave oligonucleotide probe is first synthesized with DNA and RNAsequences that are complimentary to sequences found within a PCRamplicon comprising a selected target sequence. In one embodiment, theprobe is labeled with a FRET pair, for example, a fluorescein moleculeat one end of the probe and a non-fluorescent quencher molecule at theother end.

Real-time nucleic acid amplification is then performed on a targetpolynucleotide in the presence of a thermostable nucleic acidpolymerases, a thermostable modified RNase H activity, a pair of PCRamplification primers capable of hybridizing to the targetpolynucleotide, and a labeled CataCleave oligonucleotide probe. For thedetection of a target RNA sequence, the reaction mix includes a reversetranscriptase activity for an initial cDNA synthesis step as describedherein. During the real-time PCR reaction, hybridization of the probewith the PCR amplicons forms a RNA:DNA heteroduplex that can be cleavedby an RNase H activity. Cleavage of the probe by RNase H leads to theseparation of the fluorescent donor from the fluorescent quencher andresults in the real-time increase in fluorescence of the probecorresponding to the real-time detection of the target DNA sequences inthe sample.

In certain embodiments, the real-time nucleic acid amplification permitsthe real-time detection of a single target DNA molecule in less thanabout 40 PCR amplification cycles.

Kits

The disclosure herein also provides for a kit format which comprises apackage unit having one or more reagents for the real-time detection oftarget nucleic acid sequences in a sample. The kit may also contain oneor more of the following items: buffers, instructions, and positive ornegative controls. Kits may include containers of reagents mixedtogether in suitable proportions for performing the methods describedherein. Reagent containers preferably contain reagents in unitquantities that obviate measuring steps when performing the subjectmethods.

Kits may also contain reagents for real-time PCR including, but notlimited to, a thermostable polymerases, thermostable modified RNase H,primers selected to amplify a nucleic acid target sequence and a labeledCataCleave oligonucleotide probe that anneals to the real-time PCRproduct and allows for the detection of the target nucleic acidsequences according to the methodology described herein. Kits maycomprise reagents for the detection of two or more target nucleic acidsequences. In another embodiment, the kit reagents further comprisedreagents for the extraction of genomic DNA or RNA from a biologicalsample. Kit reagents may also include reagents for reversetranscriptase-PCR analysis where applicable.

EXAMPLES

The following examples set forth methods for using the modified RNAse Henzyme composition according to the present invention. It is understoodthat the steps of the methods described in these examples are notintended to be limiting. Further objectives and advantages of thepresent invention other than those set forth above will become apparentfrom the examples which are not intended to limit the scope of thepresent invention.

Example 1 Pfu RNase HII Cleavage Assays

The qualitative cleavage activity of Pfu RNase HII was examined wherethe amount of Pfu in the reaction was held constant while theprobe:template ratio was varied. Activity was also examined where theprobe:template ratio was held constant while the amount of Pfu in thereaction was varied.

The amino acid sequence of Pfu RNase HII is SEQ ID NO: 1 and reproducedbelow.

(SEQ ID: 1)MKIGGIDEAG RGPAIGPLVV ATVVVDEKNI EKLRNIGVKD SKQLTPHERK NLFSQITSIA 60DDYKIVIVSP EEIDNRSGTM NELEVEKFAL ALNSLQIKPA LIYADAADVD ANRFASLIER 120RLNYKAKIIA EHKADAKYPV VSAASILAKV VRDEEIEKLK KQYGDFGSGY PSDPKTKKWL 180EEYYKKHNSF PPIVRRTWET VRKIEESIKA KKSQLTLDKF FKKP 224

Pfu RNase HII activity was measured as follows. Reaction mixtures eachcontaining 30 μl of 10× reaction buffer, 20 pmol of probe, varying pmolof template, and H₂O to 3004 were incubated at 64° C. for 10 minutes,then 1 μl of Pfu RNase HII was added. The reaction buffer, probe, andtemplate compositions are described in Example 2, below. Cleavage of thefluorescein-labeled probe by Pfu RNase HII was monitored by fluorescenceemission at 520 nm (upon excitation at 490 nm). Results are shown inFIG. 11A. The results in FIG. 11A show that as the probe:template ratiois increased the reaction rates increased. At a 20:20 pmol ratio thereaction is instant, meaning that the Pfu RNase HII is in great excessover the substrates. Having determined that this ratio is correctly set,experiments were performed with different concentrations of Pfu. Theresults are shown in FIG. 11B. Pfu RNase HII was diluted into 1×reaction buffer. As the Pfu RNase HII is diluted, cleavage activitydecreased. At a dilution of 1:200 the rate was linear with time.

Example 2 Reversible Formaldehyde Crosslinking of Pfu RNase HII

Pfu RNase HII was subjected to formaldehyde crosslinking using variousconcentrations of formaldehyde.

The following buffering agents were used.

Crosslinking Buffer: 20 mM HEPES, pH 7.9, 200 mM KCl, and 1 mM EDTA 2×RNase HII Storage Buffer: 100 mM Tris-HCI, pH 8.0, 200 mM NaCl, and 0.2mM EDTA

25 μL of 25 mg/mL (about 50 OD) Pfu RNase HII was diluted with 475 μL ofthe crosslinking buffer (1.25 mg/mL, about 2.5 OD). The Pfu RNase HIIwas subject to crosslinking reactions on ice under the followingconditions:

1.25 mg/mL Total Pfu Crosslinking Pfu RNase HII 13.8% Formaldehyde inVolume % Formaldehyde Conditions (ul) H20 (ul) H2O (made fresh) (ul)(ul) (Final) 1 10 8.00 0.00 18 0.00 2 10 7.75 0.25 18 0.19 3 10 7.500.50 18 0.38 4 10 7.25 0.75 18 0.58 5 10 7.00 1.00 18 0.77 6 10 6.002.00 18 1.53 7 10 4.00 4.00 18 3.07 8 10 0.00 8.00 18 6.13

Then, the reaction mixtures were incubated in 37° C. water bath for 30min. The mixtures were placed on ice and 2 μL of 2 M Tris-HCI, pH 8.0was added to each reaction mixture. After completion of the reaction,the reaction mixture was purified using G50 microspin columnspre-equilibrated with 2× RNase HII Storage Buffer, followed by dilutionwith an equal volume of glycerol for storage at −20° C. A series ofisothermal cleavage reactions were performed to test each crosslinkingcondition in the table above using the conditions described below.Reactions of type “A” did not contain target complementary to the probe.Reactions of type “C” did not contain any Pfu RNase HII.

Reaction Type A B C Reaction volume (μL): 20 20 20 Crosslinked Pfu (μL):1 1 0 10x Reaction Buffer 2 2 2 5 μM inv-CC-Probe 1 Probe (SEQ ID NO: 2)1 1 1 0.1 μM Sal Target Oligo (SEQ ID NO: 3) 0 1 1 Ultrapure H20 16 1516

1× Reaction Buffer has the following composition:

-   -   32 mM HEPES-KOH, pH 7.8    -   100 mM potassium acetate    -   4 mM magnesium acetate    -   0.11% bovine serum albumin    -   1% dimethylsulfoxide    -   4 mM MgCl2

inv-CC Probe 1 Probe had the following sequence:

(SEQ ID NO: 2) 5′-FAM-TCTGGTTGArUrUrUrCCTGATCGCA-Iowa Black FQ-3′

Sal Target Oligo had the following sequence:

(SEQ ID NO: 3) 5′-TGCGATCAGGAAATCAACCAGA-3′

SEQ ID NO: 3 is complementary to SEQ ID NO: 2 and serves as the templateto which the probe hybridizes.

RNase HII activity was measured at 50° C. without activation and at 50°C. after 95° C. activation for 15 minutes.

Cleavage of the fluorescein-labeled probe by Pfu RNase HII was monitoredby fluorescence emission at 510 nm (upon excitation at 465 nm) at theindicated assay temperature for duration of 0-30 minutes. The resultsare shown in FIG. 4. Assays in which the Pfu was not reacted withformaldehyde are referred to as “Mock-treated Pfu” and correspond to Pfucrosslinking condition number one in the table above. The results of theexperiments demonstrated that Pfu crosslinking condition number fourresulted in the greatest degree of inhibition of cleavage activity andgreatest recovery of activity after activation at 95° C. and are shownin the figures. FIG. 4A shows that reaction of Pfu with 0.75 ul of 13.8%formaldehyde resulted in the inhibition of Pfu cleavage activity similarto that seen in the absence of enzyme. Activity of the mock treated Pfusample represents baseline cleavage activity. After activation at 95°C., cleavage activity of the formaldehyde treated Pfu was restored toapproximately 50% of the mock treated level as shown in FIG. 4B. FIGS.4B and 4C demonstrate that probe cleavage did not occur in the absenceof template under the assay conditions.

Example 3 Detection of Salmonella invA Amplification usingformaldehyde-crosslinked RNase HII

Performance of formaldehyde-crosslinked Pfu RNase HII was measured usingthe Salmonella invA CataCleave™ PCR assay. Untreated Pfu RNase HII andformaldehyde-crosslinked Pfu RNase HII (Sample #4 in Example 2) weretested for their ability to function in a CATACLEAVE™ PCR assay fordetecting 5 to 5×10⁶ copies of the Salmonella invA gene target.

CATACLEAVE™ Master Mix (in μl):

Untreated Hot Star  Pfu Pfu (Sample 1) (Sample 4) Number of reactions: 99 Reaction volume (μL): 25 25 Sample volume (μL): 10 1010x I Buffer w/ 40 mM MgCl2 22.5 22.5 2 mM dNTP mix (4 mM dUTP) 9 925 μM inv-CC-Probe2 Probe* 1.8 1.8 100 μM Salmonella-F1 Primer** 1.8 1.8100 μM Sal-InvR2 Primer*** 1.8 1.8 Ultrapure H₂O 88.2 88.2Uracil DNA N-Glycosylase 0.9 0.9 RNase HII 4.5 4.5 Taq DNA Polymerase4.5 4.5 total volume of ReadyMix: 135 135 inv-CC-Probe 2 Probe:5′-FAM-CGATCAGrGrArArATCAACCAG (SEQ ID NO: 4)-Iowa Black FQ-3′,**Salmonellas-F1 primer: 5′-TCGTCATTCCATTACCTACC (SEQ ID NO: 5)-3′,***Sal-InvR2 Primer: 5′-TACTGATCGATAATGCCAGACGAA (SEQ ID NO: 6)-3′.

As can be seen from FIGS. 5A and 5B, there is no significant differencebetween the PCR efficiencies of the untreated Pfu RNase HII(“Mock-treated Pfu” in FIG. 5, sample #1 in Example 2) and theformaldehyde-crosslinked Pfu RNase HII (“Formaldehyde crosslinked Pfu”in FIG. 5, sample #4 in Example 2). Cp values were higher by about 2.5-3cycles for the formaldehyde-crosslinked RNase HII as compared with theuntreated RNase HII. The endpoint fluorescence was also lower for theamplification using formaldehyde-crosslinked RNase HII.

Example 4 Reversible Cis-Aconitylation of Pfu RNase HII

Pfu RNase HII was subjected to cis-aconitylation using variousconcentrations of cis-aconitic anhydride from 50:1 to 200:1 molar ratiosof cis-aconitic anhydride to the enzyme (see FIG. 3).

The following buffers were used for the acylation.

2× RNase HII Storage Buffer:

-   -   100 mM Tris-HCI, pH 8.0    -   200 mM NaCl    -   0.2 mM EDTA

10× Acylation Buffer

-   -   500 mM Tris-HCI, pH 7.5    -   650 mM KCl    -   10 mM EDTA

25 μL of 25 mg/mL (approximately 50 OD) Pfu RNase HII was diluted with475 μL of acylation buffer (1.25 mg/mL, approximately 2.5 OD) andacylation reactions were set up on ice. Reaction mixtures were incubatedat 4° C. for about 18 hours. Reaction mixtures were purified using G50microspin columns pre-equilibrated with 2× RNase HII Storage Buffer.

Each reaction mixture was diluted with an equal volume of glycerol forstorage at −20° C. The molar ratios of cis-aconitic anhydride and RNaseHII and other acylation conditions are shown in Table below.

1.25 mg/mL 10x 10 mg/mL cis-aconitic Total Pfu RNase HII H20 Acylationanhydride in EtOH Volume Molar Ratio (cis- Sample # (ml) (ml) Buffer(ml) (made fresh) (ml) (ml) Aconitic anhydride/RNase) 1 10 8.00 2 0.0020 0 2 10 7.62 2 0.39 20 50 3 10 7.23 2 0.77 20 100 4 10 6.46 2 1.54 20200

Then, the endonuclease activity of the cis-aconitylated Pfu RNase HIIwas determined at 50° C. with and without a heat treatment(reactivation) at 95° C. for 15 minutes. The following RNase HII assaymix was used to measure the RNase HII activity.

RNase HII Assay master Mix (1) (in μl) A B Number of reactions: 20 20Reaction volume (μL): 20 20 Sample volume (μL): 1 15x Tris-Acetate Buffer, pH 8.4 80 80 5 μm inv-CC-Probe1 Probe* 20 200.1 μM Sal Target Oligo** 0 20 Ultrapure H₂0 280 260total volume of ReadyMix: 380 380 *inv-CC-Probe1:5′-FAM-TCTGGTTGArUrUrUrCCTGATCGCA (SEQ ID NO: 2)-Iowa Black FQ-3′ **SalTarget Oligo sequence: 5′-TGCGATCAGGAAATCAACCAGA (SEQ ID NO: 3)-3′

The results are shown in FIG. 6A (without reactivation) and FIG. 6B(with reactivation). The results show that about 200:1 molar ratio ofcis-aconitic anhydride:enzyme is necessary for complete inactivation ofRNase HII activity at 50° C. All concentrations of cis-aconiticanhydride used resulted in near full reactivation of RNase HII activityafter 15 min at 95° C., when the reactivation (i.e., heating at 95° C.)was performed using Tris acetate, pH 8.4 buffer.

In order to evaluate whether a buffering agent has any impact on thereactivation, the same reactivation procedure was performed using aRNase HII assay master mix (2) which uses 1× reaction buffer(composition described in Example 2).

RNase HII Assay master Mix (2) (in μl) A B Number of reactions: 7 7Reaction volume (μL): 20 20 Sample volume (μL): 1 1 10x Reaction Bufferw/ 40 mM 14 14 5 μm inv-CC-Probe 1 Probe 7 7 0.1 μm Sal Target Oligo 0 7Ultrapure H₂O 112 105 total volume of ReadyMix: 133 133

RNase HII activity was measured at 50° C. (60 cycles) for a 30-secondhold time for each cycle.

The results (not shown) indicate that reaction buffer, which containsHEPES-KOH pH 7.8 (instead of Tris-HCl), is also capable of reactivation.

Example 5 Detection of Salmonella invA Using Acylated RNase HII

Performance of formaldehyde crosslinked Pfu RNase HII (Sample 4 inExample 2) was measured using Salmonella invA and CataCleave™ PCR assay.The master mix for amplification was as follows. The assay was performedat pH 8.4 or pH 8.7.

CATACLEAVE ™ Master Mix (in μl) pH 8.4 pH 8.7 Number of reactions: 6 6Reaction volume (μL): 25 25 Sample volume (μL): 10.5 10.55x Tris-Acetate Buffer 30 30 2 mM dNTP mix (4 mM dUTP) 6 625 μm inv-CC-Probe2 Probe* 1.2 1.2 100 μm Salmonella-F1 Primer** 1.2 1.2100 μm Sal-InvR2 Primer*** 1.2 1.2 Ultrapure H₂O 43.8 43.8Uracil DNA N-Glycosylase 0.6 0.6 Taq DNA Polymerase 3 3total volume of ReadyMix: 87 87 *inv-CC-Probe 2 Probe:5′-FAM-CGATCAGrGrArArATCAACCAG (SEQ ID NO: 4)-Iowa Black FQ-3′**Salmonella-F1 primer: 5′-TCGTCATTCCATTACCTACC (SEQ ID NO: 5)-3′,***Sal-InvR2 Primer: 5′-TACTGATCGATAATGCCAGACGAA (SEQ ID NO: 6)-3′.

The results of FIG. 7A (at pH 8.4) and FIG. 7B (pH 8.7) demonstrate thatusing either Tris-acetate buffer (pH 8.4 or 8.7) is adequate to get nearfull re-activation of the cis-aconitylated Pfu RNase HII in theSalmonella invA CATACLEAVE qPCR assay (with a 15 min 95° C. heattreatment).

Also, about 200:1 molar ratio of cis-aconitic anhydride to enzyme seemsto be necessary and sufficient for creating a hot-start Pfu RNase HIIthat functions well in amplification and has minimal RNase H activitywithout heat activation.

Example 6 One-Step RT PCR Amplification of HIV-1

The reversibly modified RNase HII as described and prepared above may beparticularly suitable for use in one-step RT PCR amplification of viralRNA targets. The modified, inactive RNase HII does not cleave targetribonucleic acid molecules in a sample and it allows reversetranscriptase to produce viral cDNA molecules from the RNA nucleicacids.

In the instant exemplary embodiment, the modified RNase HII is used todetect a HIV-1 target RNA. Sample #4 of Example 2 (composition 2) andsample #4 of Example 3 (composition 3) were used as modified RNase HII.For comparison, unmodified Pfu RNase HII were used (composition 1).

1 X Tris pH 8.7 Buffer 6  10 mM Tris base pH 8.7 (w/ acetic acid) 50 mM KOAc 2.5 mM MgOAc   1 mM DTTComposition 1. One step RT-PCR Tris 8.7 buffer 6 (unmodified Pfu RNase HII) (in μl)     1 ul HIV-1_F11-JO primer* 7.5 uM      1 ulHIV-1_R6-JO primer** 7.5 uM      1 ul HIV-1_CCProbe5*** 10 uM      1 uldNTP 10 mM each      5 ul 5 x Tris 8.7 buffer 2****      1 ulRT-PCR enzyme mix      1 ul Pfu RNase HII     13 ul H2Oadd 24 ul to 1 ul RNA templateComposition 2. RT-PCR Tris 8.7 buffer 6 with modified RNase HII of Example 2(Sample 4) (in μl)      1 ul HIV-1_F11-JO primer* 7.5 uM      1 ulHIV-1_R6-JO primer** 7.5 uM      1 ul HIV-1_CCProbe5*** 10 uM      1 uldNTP 10 mM each      5 ul 5 x Tris 8.7 buffer 2****      1 ulRT-PCR enzyme mix      1 ul Pfu RNase HII (formaldehyde)     13 ul H2Oadd 24 ul to 1 ul RNA templateComposition 3. One step RT-PCR Tris 8.7 buffer 6 with modified RNase HII ofExample 3 (Sample 4)      1 ul HIV-1_F11-JO primer* 7.5 uM      1 ulHIV-1_R6-JO primer** 7.5 uM      1 ul HIV-1_CCProbe5*** 10 uM     1 ul            dNTP 10 mM each      5 ul 5 x Tris 8.7 buffer 2****     1 ul RT-PCR enzyme mix      1 ulPfu RNase HII (cis-aconitic anhydride)     13 ul H2Oadd 24 ul to 1 ul RNA template *HIV-1-F11-JO: 5′-CCAAGGGGAA GTGACATAGCAGGAACTACT-3′, (SEQ ID NO: 7) **HIV-1_R7-JO:5′-CTGACGACAGGGCTATACATTCTTACTATTT-3′, (SEQ ID NO: 8) ***HIV-1_CCProbe:5′-FAM-TACCCTTCAGrGrArArCAAATAGGATGGAT-IABlk_FQ-3′, (SEQ ID NO: 9)****Buffer 2: Tris-Acetate pH 8.7, 50 mM KOAc, 2.5 mM MgOAc, and 1 mMDTT. RT-PCR conditions were as follows: 50° C. 30 min 95° C. 15 min 50cycles of 95° C. 30 sec, 60° C. 30 sec, and 72° C. 60 sec.

The endonuclease activity of RNase HII was measured by following theprocedure described in Example 2.

The results are shown in FIG. 8, each show the fluorescence determinedon the RT-PCR products using the composition 1, composition 2, andcomposition 3, respectively. As shown in FIG. 8, the acylated RNase HIIshowed a steeper curve than that of formaldehyde-crosslinked RNase HII,probably because the acylated RNase HII is reactivated more fully underthe conditions used in the Example. Full or complete recovery ofactivity of formaldehyde treated RNase HII by high temperatureincubation is lower than that seen with acylated treated enzyme bychanges in pH for equivalent samples.

Example 7 Sensitivity of Reversibly Acylated RNase HII

In order to determine the sensitivity of the modified RNase HII, thesame procedure described in Example 6 was followed using the acylatedRNase HII (sample #4 of Example 3), except different concentrations ofHIV-1 target RNA were present in the amplification composition.

The results are shown in FIG. 9. As can be seen in FIG. 9, the one-stepRT PCR containing an acylated Pfu RNase H could detect as few as 10input copies of HIV-1 genomic RNA. No amplification was observed in thenegative control. Also, the reactions showed concentration dependence(not shown).

Example 8 Comparison Between Reversibly Acylated RNase HII andUnmodified RNase HII

1 X Tris pH 8.7 Buffer 6  10 mM Tris base pH 8.7 (w/ acetic acid) 50 mM KOAc 2.5 mM MgOAc   1 mM DTT One step RT-PCR CataCleave buffer 6   0.5 ul Salmonella-Fl primer*    0.5 ul Sal-invR2 primer **      1 ulinv_CCProbe1 5 uM***      1 ul dNTP 10 mM each    2.5 ul5 x Tris 8.7 buffer 6****      1 ul RT-PCR enzyme mix    0.5 ulPfu RNase HII (cis-aconitic anhydride)     17 ul H2Oadd 24 ul to 1 ul RNA template *Salmonella-F1 primer:5′-TCGTCATTCCATTACCTACC-3′, (SEQ ID NO: 5) ** Sal-invR2 primer:5′-TACTGATCGATAATGCCAGACGAA-3′, (SEQ ID NO: 6) ***inv_CCProbe1:5′-FAM-TCTGGTTGArUrUrUrCCTGATCGCA-3IAB1k_FQ-3′, (SEQ ID NO: 2)****Buffer 6: Tris-Acetate pH8.7, 50 mM KOAc, 2.5 mM MgOAc, 1 mM DTT.RT-PCR conditions were as follows: 50° C. 30 min 95° C. 15 min 50 cyclesof 95° C. 30 sec, 60° C. 30 sec, and 72° C. 60 sec.

Salmonella invA RNA of about 1500 nucleotides was synthesized using aT7/polymerase system. The RNA was quantified and standardized by copynumbers into pre-aliquoted dilutions of, 10⁶, 10⁵, 10⁴, 10³, 10², and 10copies/W. These dilutions were stored at about −80° C. until use.

One-step RT-PCR amplification compositions (as shown above “one stepReverse Transcriptase-PCR CATACLEAVE™ buffer 6”) each containing 10⁶,10⁵, 10⁴, 10³, 10², and 10 copies invA RNA/μl were subjected to RT-PCRunder the following conditions, and then fluorescence (465-510 nm) ofthe resulting amplification product was measured. As an acylated PfuRNase HII, sample #4 of Example 3 was employed. For a comparison, thesame amplification composition, which contains an unmodified Pfu RNaseHII, rather than the acylated Pfu RNase HII of Example 3 (Sample #4),was used. Results are shown in FIG. 10.

As can be seen in FIG. 10, the composition containing the acylated RNaseHII was able to detect as few as 10 copies of Salmonella RNA targetnucleic acid having about 1500 nucleotides. When untreated Pfu RNase HIIwas used, a large Cp shift (about 10 cycles) and loss of sensitivity(1-2 orders of magnitude) were observed. This experiment demonstratedthat if the RT reaction is performed in the presence of active RNaseHII, that sensitivity is decreased probably due to degradation of theRNA template. This is manifested as a marked increase in Cp values forthe real-time reaction.

Any patent, patent application, publication, or other disclosurematerial identified in the specification is hereby incorporated byreference herein in its entirety. Any material, or portion thereof, thatis the to be incorporated by reference herein, but which conflicts withexisting definitions, statements, or other disclosure material set forthherein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.

What is claimed:
 1. A method of amplifying a target sequence in asample, comprising providing an amplification reaction mixturecomprising: a sample comprising a target DNA sequence; a hot startenzyme composition, comprising DNA polymerase and an enzyme having aninducible RNase H activity, wherein said enzyme is reversibly modifiedby acylation of an amino acid residue of said enzyme or withformaldehyde; a first primer comprising a sequence complementary to the5′ end of the target nucleic acid sequence; a second primer comprising asequence complementary to the 3′ end of the target nucleic acidsequence; and a probe which contains a detectable label and comprises acleavage sequence of the RNase H; subjecting the amplification reactioncomposition to at least one amplification reaction to form at least oneamplification product, and measuring the detectable label of theresulting amplification product, wherein the amplification reactioncomprises a step of heating the reaction composition to a temperature ofabout 90° C.
 2. The method of claim 1, wherein the step of heating thereaction composition is conducted at about 95° C.
 3. The method of claim1, wherein the probe is an oligonucleotide comprising one or more DNAsequence portions and a RNA sequence portion, wherein the RNA portion isdisposed between two DNA sequences in a way that 3′ end and 5′ end ofthe RNA sequence is coupled to each of the two DNA sequences.
 4. Themethod of claim 1, wherein said RNase H activity is heat-inducible. 5.The method of claim 1, wherein said RNase H activity is inducible bymodifying the pH of a solution containing the enzyme.
 6. The method ofclaim 1, wherein said amino acid is lysine.
 7. The method of claim 1,wherein the formaldehyde is a solution containing formaldehyde atconcentration of about 0.2-1% (w/v).
 8. A method for detecting a targetribonucleic acid sequence in a sample, comprising providing anamplification reaction mixture comprising: a sample containing a targetRNA sequence, a hot start enzyme composition, comprising reversetranscriptase, DNA polymerase and an enzyme having an inducible RNase Hactivity, wherein said enzyme is reversibly modified by acylation of anamino acid residue of said enzyme or with formaldehyde; a first primercomprising a sequence complementary to the 5′ end of the target nucleicacid sequence; a second primer comprising a sequence complementary tothe 3′ end of the target nucleic acid sequence; and a probe sequencewhich contains a detectable label and comprises a cleavage sequence ofthe RNase H; initiating reverse transcription of the target RNA to forma RNA:cDNA duplex; heating the reaction composition to a temperature ofabout 90° C. or higher thereby activating the inducible RNase H activityto degrade the RNA moiety of the RNA:cDNA duplex; initiating at leastone amplification reaction to form at least one amplification product,and measuring the detectable label of the resulting amplificationproducts.
 9. The method of claim 8, wherein the probe is anoligonucleotide comprising one or more DNA sequence portions and a RNAsequence portion, wherein the RNA portion is disposed between two DNAsequences in a way that 3′ end and 5′ end of the RNA sequence is coupledto each of the two DNA sequences.
 10. The method of claim 8, wherein thetarget ribonucleic acid sequence is a retrovirus.
 11. The method ofclaim 8, wherein the step of heating the reaction composition isconducted at about 95° C.
 12. The method of claim 8, wherein said RNaseH activity is heat-inducible.
 13. The method of claim 8, wherein saidRNase H activity is inducible by modifying the pH of a solutioncontaining the enzyme.
 14. The method of claim 8, wherein said aminoacid is lysine.
 15. The method of claim 8, wherein the formaldehyde is asolution containing formaldehyde at concentration of about 0.2-1% (w/v).16. A method for detecting target ribonucleic acid sequences in aplurality of samples, comprising providing a microarray having aplurality of amplification reaction mixtures each comprising: a samplecontaining a target RNA sequence, a hot start enzyme composition,comprising reverse transcriptase, DNA polymerase and an enzyme having aninducible RNase H activity, wherein said enzyme is reversibly modifiedby acylation of an amino acid residue of said enzyme or withformaldehyde; a first primer comprising a sequence complementary to the5′ end of the target nucleic acid sequence; a second primer comprising asequence complementary to the 3′ end of the target nucleic acidsequence; and a probe sequence which contains a detectable label andcomprises a cleavage sequence of the RNase H; and for each amplificationreaction composition in the microarray: initiating reverse transcriptionof the target RNA to form a RNA:cDNA duplex; heating the reactioncomposition to a temperature of about 90° C. or higher therebyactivating the inducible RNase H activity to degrade the RNA moiety ofthe RNA:cDNA duplex; initiating at least one amplification reaction toform at least one amplification product, and measuring the detectablelabel of the resulting amplification product.
 17. The method of claim16, wherein the target ribonucleic acid sequence is a retrovirus. 18.The method of claim 16, wherein the step of heating the reactioncomposition is conducted at about 95° C.
 19. The method of claim 16,wherein said RNase H activity is heat-inducible.
 20. The method of claim16, wherein said RNase H activity is inducible by modifying the pH of asolution containing the enzyme.